Basic Technique in Haematology

Dr Eow Geok Im MD(UKM), MPath(Haemtology)(UM)

 Aim
Brief background on basic laboratory techniques in clinical hematology, with emphasis on common manual and automated hematological laboratory tests

Introduction
Why we need laboratory tests?
Diagnosis, understanding pathophysiology, monitoring of treatment

Haematological tests
Is the marrow producing sufficient no of mature cells? Are they qualitatively normal?

 What are the common tests?

Full Blood Count
Red cells RBC,PCV, Hb, red cell indices, reticulocytes White cells WBC, leukocyte differential Platelets

Morphological examination
Blood film preparation stain

 Collection
Anticoagulant
Ethylenediamine tetra-acetic acid (EDTA), dipotassium salt 1.50.25mg/ml Excess may cause
Decrease PCV, increased MCHC Increase MCV Plts swell and disintegrate

Venous or capillary
Capillary infants <1 year; free flowing, arteriolar PCV, RBC, Hb, WBC(neutrophils, monocytes) higher in capillary plts are higher in venous

Pre-analytical variables
Correct amount Effects of storage morphology, quantitative
Red cell- crenation White cell- nuclei stain homogenous with ragged cytoplamic margin

Sample homogeneity Inherent factors eg. Age, sex, genetic background

 Reference ranges or normal values?

Normal value can be abnormal Statistical procedures
Normal distribution Mean 2 SD

 Manual
Low cost Labor intensive Lower precision as standard

Automation
High capital Rapid performance Less labor Precise Need calibration and maintenance

Red cells
Quantitative measurement
Hb RBC HCT/PCV RBCs indices (sizes and haemoglobin content of RBC) Reticulocytes

Haemoglobin
Erythrocytes content
Mixture of haemoglobin, oxyhaemoglobin, carboxy haemoglobin, methaemoglobin and other Properties: Colour, combination with O2/CO, iron content

Principle of measurement (Cyanmethaemoglobin method)

RC are lysed Hb variants (except sulphaemoglobin) are converted to the stable compound cyanmethaemoglobin Quantitation by absorption at 540nm (spectro)

Hb(Fe2+)

K3Fe(CN)6

Hi(Fe3+)

KCN

HiCN

Haemoglobin

Clinical implication
The iron-containing protein attached to RBCs that transports oxygen from the lungs to the rest of the body Diagnosis of anaemia

Other method of measurement

Oxyhaemoglobin method, direct reading haemoglobinometry

ICSH recommendation
Cyanmethaemoglobin method Very broad absorption peak (535-545nm) Allow direct comparison with HiCN std

Quality control
Commercial controls, proficiency testing, control to be run with each batch of specimens

Haemoglobin

Problems and error

Cyanide potential hazard, alternative: sodium azide, sodium lauryl sulphate (no stable std); HiCN reagent is sensitive to light Technical error pipetting, cuvette, deteriorated reagent Turbidity in mixture will cause falsely elevated value (hyperleucocytosis, lipemia, abnormal plasma protein) RC which are relative resistance to lysis (HbS, HbC)

Assignment / Practical
Reagent used, procedure, standard curve preparation, calculation of Hb concentration, Hb reference ranges

Haemoglobin

Automation
Direct measurement Modification of manual HiCN method
Concentration of reagent, temperature and pH of reaction Addition of non-ionic detergent to ensure rapid cell lysis, reduce turbidity Measurement at set interval before the reaction is completed Utilization of non cyanide reagent

Packed cell volume or Haematocrit

The ratio of volume occupied by the packed red blood cells to the volume of whole blood Use for
Screening for anaemia Estimation of red cell indices Calibration of automated blood counter

Mode of measurement
Manual: centrifuged microhaematocrit (PCV) Automated: Generation of electrical pulses (Hct)

PCV

Centrifuged Microhaematocrit
A small amount of whole blood is centrifuged to determine maximum packing of erythrocytes Error
Technical: failure to mix, EDTA in excess, improper sealing, inadequate centrifugation Physiologic: plasma trapping increased in hypochromic anaemia, spherocytes; venous blood 2% higher

pcv

Automated Hct
Passage of a cell thro the aperture of an impedance counter or thro the bean of light in a light scattering instrument lead to the generation of an electrical impulse
No of pulses = count

Average pulse height = volume

Summation of height = PCV

RBC count
Manual counting
Counting red cells microscopically in diluted sample of blood contained in a counting chamber Obsolete, time-consuming, inaccurate

Automation
Aperture impedance Light-scattering technology

 Aperture impedance counting

Beckman-Coulter, Sysmex, Abbott, Roche Rbcs are poor conductors of electricity Dilute in a buffered electrolyte solution
cells through an aperture, causes a change in electrical resistance, this pulse is detected and amplified by the instrument.
amplitude of the pulse is proportional to cell size

 Light scatter
Diluted cell suspension flow in a single file A light source in front of the aperture Scatter light is detected by photomultipier or photodiode Convert into electrical impulses

RBC count

Threshold setting
Lower threshold to discriminate plt but include microcytic RBCs Multichannel instrument threshold are precalibrated or automatically adjusted

Errors
Inaccuracy due to coincidence or recirculation
Sheath flow or hydrodynamic focusing - cells passing in single file Sweep flow directed stream of diluents

Faulty maintenance inaccurate aspiration vol Threshold setting, appropriate diluents Sample eg.cold agglutinin

Red cell indices

MCH, MCV, MCHC etc. Derivative
Mean cell volume (fl)
PCV(l/l) x 1000 RBC(1012/l)

Mean cell haemoglobin (pg)

Hb (g/dl) x 10 RBC(1012/l)

Mean cell haemoglobin concentration (g/l)

Hb (g/dl) PCV (l/l)

 Indices provided by automated system are of considerable clinical importance

Classification of anaemia

RBC indices

MCV can be a direct measurement using automated system

Factitious elevation in hyperosmolarity, cold agglutinin
Pulse height average = MCV No of impulses = RBC Summation of pulse height = PCV

Other RBC indices

Provided by the automated system
Red cell distribution width (RDW)
Volume distribution histogram
Degree of anisocytosis Derived from pulse height analysis CV(%) or SD(fl)

Other RBC indices

Cellular haemoglobin concentration mean (CHCM)

Direct measurement using light scattering at different angle in Bayer-Technicon To replaced the role of MCHC. Sensitivity to iron deficiency has improved. As a internal quality control. If all measurement accurate, MCHC=CHCM

Haemoglobin distribution width (HDW)

Bayer-Technicon, determine Hb concentration on individual cell Degree of variation in red cell haemoglobinization

Reticulocyte count
Juvenile red cells - remnants of the ribosomal ribonucleic acid Used to assess bone marrow erythropoietic activity

reticulocytes

Procedure:
Stain: NMB, brilliant cresyl blue, purified azure B Incubate at 37C for 15 min Make films and examine microscopically Count using x 100 oil-immersion Technique

Subjective with low precision and accuracy

Well spread, well stain, observer, quality of microscope

Absolute reticulocyte count

reticulocytes

Automation:
Stain: auramine O(sysmex), thiazole orange(ABX), CD4K 530(Abbott), Oxaxine 750(bayer-technicon), NMB(Beckmancoulter) fluorescent cell enumerated using flow cytometry Better precision (more cells counted, better recognicing of late reticulocytes) Error: inclusion of WBC and plt, Howell-Jolly bodies, malarial parasites Assessment of reticulocytes maturity: more RNA, fluoresce stronger
Sign of engraftment, clinical response to treatment (SAA), predicting optimal time for stem cell harvesting

 Summary
Manual techniques
Red cells
HB, PCV, RBC, reticulocyte Principle of measurement, error, clinical application Derivative: MCV, MCH, MCHC

Automation
Principle New parameters (RDW, CHCM, HDW, % of hypochromic cells)

White Cells and Platelet

Manual cell count
Acceptable alternative to electronic counter Simple instrument: microscopes, Neubauer counting chamber Error:
Technical Technician mixing, faulty in filling of chamber, careless counting Instrument chamber, pippete, microscope Inherent Uneven distribuition of cells in counting chamber

White cell and platelets

Automation
Impedance or light scattering WBC count
Red cells are lysed, residual particles are counted Threshold are set for WBC to exclude plt Error for WBC: giant plt, nRBC, white cell agglutination

Platelet count
Counted in WB suing electrical electro-optical detection Threshold is set to seperate red cells, debris and electronic noise New plts parameter: MPV, PDW

Differential Counts
Manual
Visual examination of blood films Misdistribution of various cells

BODY

TAIL

Differential count

Counting the differential

From head to tail:
Error: maldsitribution, misinterpretation (thick film)

Battlement method

Assign the leucocytes, express in % (5 part or more) ??nRBC, corrected WBC how many cells to count? Precision vs practicality No best counting method in badly made film

Automated Differential
More precise but sometimes inaccurate Flow cytometry principle Diluted WB, RC lysed, WC categorised into 3 or 5 part diff Single channel or 2/ more channel Based on
Volume Physical characteristics Activity of cellular enzyme

Auto diff

3 part
Single channel Based on volume of various cells 3 categories
Granulocytes/LC Lymphocytes/SC Monocytes/MNC

5 part or more
2 or more channel Cell volume and other characteristics 5 categories
Neutrophils Eosinophils Basophils Lymphocytes Monocytes

Eosinophil/basophils are included in MNC

Other may have LIC, AL..

Auto diff

Instrument principle
Light scattering and absorbance Impedance measurement with low and high frequency electromagnetic current or radiofrequency current Cytochemical reaction

Analysis
2 parameter or more complex Cells are divided into cluster Threshold (fixed and variable)
Abbot Cell-Dyne

sysmex

 Automated vs manual
More precise Accuracy less impressive: unusually cell, aging sample

Main function of automation

Performing differential count on normal Flagging abnormal sample

Graphical display
Histogram of red cell Histogram of plt sizes Scatter plot of DC

Peripheral Blood Films

Sample: from fresh blood or EDTA-anticoagulated sample Method:
Manual: clean glass slide, spreader Automated

Labelling: name, date Fixing: immediately with methyl alcohol Staining:

automated Manual: MGG, standardized Romanowsky stain etc

Mounting
Xylol or cedarwood oil

PBF

Examination of blood films

Red cells: sizes and shape, haemoglobin, invlusion and other abnormalities White cells: quantity, differential count, abnormal morphology, abnormal granules Platelets: quantity, morphology

Platelet morphology

Platelet morphology

Neutrophils

Eosinophils, basophils, monocytes

Lymphocytes

Red cell morphology

What are the abnormalities?

 Automated image analyser

Examination of peripheral blood films using automated microscopy; evaluation of Diffmaster Octavia and Cellavision DM96.
J Clin Pathol. 2007 Jan;60(1):72-9.

References
Dacie & lewis Practical haematology Blood cells a practical guide. Barbara bain Clinical haematology. Principles, procedures, correlation Lippincott Esssential haematology AV Hoffbrand, blackwell publishing